Use of phenol substituted diphosphonates as antineoplastic agents

ABSTRACT

A method of treating or preventing neoplastic disease and a method of preventing transformation of a normal cell into a tumor cell by mutated ras activities, utilizing a compound of formula I are disclose. ##STR1##

The present invention relates to antineoplastic agents, and inparticular to the use of phenol substituted gem-diphosphonatederivatives in the treatment of neoplastic diseases. More specifically,the present invention provides the use of certain phenol substitutedgem-diphosphonate derivatives for the preparation of pharmaceuticalcompositions useful in the treatment and prevention of cancers andmetastasis and in particular useful in the treatment and prevention ofras oncogene dependent cancers and metastatic invasions.

The majority of existing anticancer drugs are cytotoxic compounds whichlack specificity for killing tumor cells and therefore also affectnormal cells, resulting in toxic side effects. There remains therefore aneed for the development of more specific agents acting on the cellsignalling pathways leading to the inhibition of cancer cellproliferation without affecting normal cell proliferation (OncolyticDrugs, J. R. Prous, The Year's Drug News, 1994 edition, p.459 and RasOncogene Directed Approaches in Cancer Chemotherapy, G. Bolton et. al.,Annual Reports in Medicinal Chemistry 1994; 29: 165-174).

Mutations of the ras oncogene have been shown to be present in a widevariety of human tumors and may contribute to as many as one-fifth ofall human cancers. Specifically, it is found in more than fifty percentof colon and ninety percent of pancreatic carcinomas. Ras mutations aretherefore considered to play a key role in triggering cancer formationand development (J. L. Bos, Cancer Res. 1989;49:4682-4689). It has alsobeen established that the mutated forms of ras protein are present onlyin tumors and not in normal tissues of cancer patients. Blocking theactivity of ras mutations to transform normal cells into cancer cells aswell as to further promote the development of cancer cells and tumors istherefore an attractive therapeutic target.

The U.S. Pat. No. 5,043,330 (1991) corresponding to the European PatentNo. 0,339,237 discloses the synthesis of a class of phenol substitutedgem-diphosphonate derivatives and their utility as lipid loweringagents, for example in the treatment of cardiovascular diseases.

The present applicants have now found that diphosphonates of the typedisclosed in U.S. Pat. No. 5,043,330 are surprisingly effective forinhibiting specifically the proliferation as well as inducing apoptosisin cancer cells without being cytotoxic to normal cells.

Accordingly, in one aspect, the invention provides the use of a compoundfor the manufacture of a medicament for the treatment of neoplasticdiseases, said compound having the following formula (I): ##STR2##where: Z¹, Z², Z³ and Z⁴ are identical or different and are

OR where R is H, a straight, branched or cyclic alkyl group comprisingfrom 1 to 8 carbon atoms,

OM where M is a cation,

NR₂ where R has the same meaning as defined above,

Z¹, Z² and Z³, Z⁴ may form an alkylidenedioxy ring comprising 2 to 8carbon atoms,

X¹ and X² are identical or different and are H, a halogen atom, astraight, branched or cyclic alkyl or alkoxy group from 1 to 8 carbonatoms,

X³ is H, an alkyl group R¹ from 1 to 4 carbon atoms, an acyl groupC(O)R¹, a carbamyl group C(O)NHR¹ where R¹ is described as above, X³ Oand one of the two other substituents X¹ or X² may form analkylidenedioxy ring comprising from 1 to 4 carbon atoms,

A is --CH═CH--CH₂ --, --(CH₂)_(n) --, --O(CH₂)_(n) --, --S--, --SO₂ --,--S(CH₂)_(n) --, --SO₂ (CH₂)_(n), where n is an integer from 1 to 7, ortogether with B forms an alkylidene group of the formula (CH═CH)_(k)--(CH₂)_(d) --CH═ where k is zero or 1 and d is an integer from zero to4,

B is H, an alkyl group from 1 to 4 carbon atoms,

t is zero or 1, with the proviso that t is zero only when A is(CH═CH)_(k) --(CH₂)_(d) --CH═ where k and d are as described above.

The compounds of formula (I) can exist as salts and references to thecompounds of formula (I) hereinafter include the salt forms of thecompounds, unless the context indicates otherwise. Examples of salts arecompounds of formula (I) wherein one or more of the groups Z¹, Z², Z³and Z⁴ are constituted by the group OM where M is an alkaline oralkaline earth metal ion or an ammonium group NR₄ where R has the samemeaning as defined above.

In another aspect the invention provides the use of a compound of theformula (I) as hereinbefore defined for the manufacture of a medicamentfor treating solid tumors, for example colon, pancreas, thyroid, lung,breast, head and neck tumors.

In another aspect the invention provides the use of a compound of theformula (I) as hereinbefore defined for the manufacture of a medicamentfor treating tumors of the hemopoietic and immune system, for examplelymphomas and leukemias.

In still another aspect the invention provides the use of a compound ofthe formula (I) as hereinbefore defined for the manufacture of amedicament for treating patients with metastasis of primary tumors.

In a further aspect, the invention provides the use of a compound offormula (I) as hereinbefore defined for the manufacture of a medicamentfor preventing the transformation of normal cells or inhibitingmetastatic invasion of normal tissues by cancer cells.

In a still further aspect, the invention provides a method of treatmentof neoplastic diseases or prevention of cancer metastasis, and inparticular ras-dependent cancers, which method comprises administeringto a patient suffering from said cancer or the potential of cancerdevelopment, an effective therapeutic amount of a compound of theformula (I) as hereinbefore defined.

The invention also includes within its scope a method for selectivelyeradicating cancer cells which comprises treating a mixture of cancercells and normal cells from a patient in an ex vivo manner with acompound of the formula (I), further to which the cells are reintroducedinto the patient. Thus, for example, in accordance with this method,blood, plasma or other fluids can be drawn off from the patient, treatedwith the compounds of formula (I) ex vivo, and then reintroduced intothe patient.

In the compounds of formula (I), examples of groups Z¹, Z², Z³ and Z⁴include hydroxy, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butyloxy,sec-butyloxy and tert-butyloxy. A preferred group is the isopropyloxygroup.

It is presently preferred that the groups Z¹, Z², Z³ and Z⁴ areidentical and in a particularly preferred embodiment of the inventionZ¹, Z², Z³ and Z⁴ are all isopropyloxy.

Examples of groups X¹ and X² include hydrogen, straight or branchedalkyl groups and alkoxy groups having from 1 to 5 carbon atoms, moreparticularly from 1 to 4 carbon atoms. Preferred groups X¹ and X² aremethyl, ethyl, n-propyl, isopropyl, sec-butyl, tert-butyl, methoxy andethoxy groups, a particularly preferred group being tert-butyl.

Examples of groups X³ include hydrogen, C₁₋₄ alkyl and C₁₋₄ alkanoyl,hydrogen being particularly preferred at present.

The compounds of formula (I) include the phenol substitutedalkylidenediphosphonates (Ia) and the phenol substitutedalkenylidenediphosphonates (Ib). ##STR3## where X¹, X², X³, A, B, k, d,Z¹, Z², Z³ and Z⁴ are as described above.

Compounds of structure (Ia) include, for example, those in which:

X¹ and X² are identical or different and are alkyl groups from 1 to 8carbon atoms,

X³ is hydrogen,

A is CH═CH--CH₂, (CH₂)_(n), S, SO₂, S--(CH₂)_(n), SO₂ --(CH₂)_(n), wheren is 1-7,

B is hydrogen or a C₁ -C₄ alkyl group,

Z¹, Z², Z³ and Z⁴ are identical or different and are OH, alkoxy groupsof 1 to 8 carbon atoms or one or both of the pairs Z¹, Z² and Z³, Z⁴ arean alkylidenedioxy group of 2 to 8 carbon atoms.

Compounds of structure (Ib) include, for example, those in which

X¹ and X² are identical or different and are alkyl groups from 1 to 8carbon atoms,

X³ is hydrogen,

k is zero or 1 and d is zero to 4,

Z¹, Z², Z³, Z⁴ identical or different are OH, alkoxy groups of 1 to 8carbon atoms or one or both of the pairs Z¹, Z² and Z³, Z⁴ are analkylidenedioxy group of 2 to 8 carbon atoms.

Particular examples of compounds of formula (I) for use in the presentinvention include the compounds in Tables 1 a and 1 b.

This invention provides a new use of gem-diphosphonates of formula (I)for the treatment of neoplastic diseases and prevention of cancers andmetastasis, and more particularly those which are ras dependent. In aparticular preferred embodiment, it provides a new use of Compound 1 offormula (I), wherein X¹ and X² are both tert-butyl respectively at the3- and 5- positions, X³ is H at the 4-position, A is CH₂, B is H, t is 1and Z¹, Z², Z³, Z⁴ all are iso-propyloxy for the preparation ofpharmaceutical compositions useful for the treatment of cancers. SaidCompound 1 has the following structure, formula and physicochemicalproperties: ##STR4## Tetraisopropyl2-(3,5-di-tert-butyl4-hydroxyphenyl)-ethylidene-1,1-diphosphonate, C₂₈H₅₂ O₇ P₂, mp=104-105° C.

The compounds of the invention can be prepared according to the methodsdescribed in EP 0 339 237 A, the disclosure of which is incorporated byreference herein, or by methods analogous thereto.

Some of the analogs are novel. Accordingly, in a further aspect, theinvention provides a novel compound selected from:

tetraisopropyl 2-(3,5-diisopropyl-4-hydroxyphenyl)-ethenylidene-1,1-diphosphonate,

tetraisopropyl2-(3,5-diisopropyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate,

tetraisopropyl 2-(3,4,5-trimethoxyphenyl )-ethenylidene-1,1-diphosphonate,

tetraisopropyl 2-(3,4,5-trimethoxyphenyl)-ethylidene-1,1-diphosphonate,

tetraisopropyl 2-(3-tert-butyl4-hydroxy-5-methylphenyl)-ethenylidene-1,1-diphosphonate,

tetraisopropyl2-(3-tert-butyl-4-hydroxy-5-methylphenyl)-ethylidene-1,1-diphosphonate,

tetraisopropyl2-(3-ethoxy4-hydroxyphenyl)-ethenylidene-1,1-diphosphonate,

tetraisopropyl2-(3-ethoxy-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate,

tetraethyl2-(3,5-di-tert-butyl-4-methoxyphenyl)-ethenylidene-1,1-diphosphonate and

tetraisopropyl1-(3,5-di-tert-butyl-4-hydroxyphenyl)butylidene-2,2-diphosphonate.

The compounds of formula (I) can be administered orally, or by deliveryacross another mucosal surface (for example across the nasal, buccal,bronchial or rectal mucosa), transdermally, or by injection (for exampleintradermal, intraperitoneal, intravenous or intramuscular injection).

When the compounds are intended for oral administration, they can beformulated, for example, as tablets, capsules, granules, pills, dragees,lozenges, powders, solutions, emulsions, syrups, suspensions, or anyother pharmaceutical form suitable for oral administration. Oral dosageforms can, if desired, be coated with one or more release delayingcoatings to allow the release of the active compound to be controlled ortargeted at a particular part of the enteric tract.

Tablets and other solid or liquid oral dosage forms can be prepared instandard fashion from the compounds of formula (I) and apharmaceutically acceptable solubilizer, diluent or carrier. Examples ofsolubilizers, diluents or carriers include sugars such as lactose,starches, cellulose and its derivatives, powdered tracaganth, malt,gelatin, talc, stearic acid, magnesium stearate, calcium sulfate,vegetable oils, polyols such as glycerol, propyleneglycol andpolyethyleneglycols, alginic acids and alginates, agar, pyrogen freewater, isotonic saline, phosphate buffered solutions, and optionallyother pharmaceutical excipients such as disintegrants, lubricants,wetting agents such as sodium lauryl sulfate, coloring agents, flavoringagents and preservatives, etc.

Capsules can be of the hard or soft variety and can contain the activecompound in solid, liquid or semisolid form. Typically such capsules areformed from gelatine or an equivalent substance and can be coated oruncoated. If it is desired to delay the release of the active compounduntil the capsule has passed through the stomach and into the intestine,the capsule can be provided with a pH sensitive coating adapted todissolve at the pH found in the duodenum or ileum. Examples of suchcoatings include the Eudragits, the uses of which are well known.

Formulations for injection will usually be made up of the appropriatesolubilizers such as detergents which may also include compounds andexcipients such as buffering agents to provide an isotonic solutionhaving the correct physiological pH. The injectable solutions aretypically pyrogen-free and can be provided in sealed vials or ampoulescontaining a unit dose of compound.

A unit dosage form of the compounds of the invention typically willcontain from 0.1% to 99% by weight of the active substance, more usuallyfrom 5% to 75% of the active substance. By way of example, a unit dosageform can contain from 1 mg to 1 g of the compound, more usually from 10mg to 500 mg, for example between 50 mg and 400 mg, and typically indoses of 100 mg to 200 mg.

The compounds of the invention will be administered in amounts which areeffective to provide the desired therapeutic effect. The concentrationsnecessary to provide the desired therapeutic effect will vary accordingto among other things the precise nature of the disease, the size,weight and age of the patient and the severity of the disease.

The doses administered will preferably be non-toxic to the patient,although in certain circumstances the severity of the disease undertreatment may necessitate administering an amount of compound whichcauses some signs of toxicity.

Typically, the compounds of the invention will be administered inamounts in the range 0.01 mg/kg to 100 mg/kg bodyweight, more preferably0.1 mg/kg to 10 mg/kg bodyweight and particularly 1 mg/kg to 5 mg/kgbodyweight. For an average human of 70 kg weight, a typical daily dosageof the compounds of the invention would be in the range of 70 mg to 700mg. Such a dosage can be administered, for example from two to fourtimes daily. Ultimately however, the size of the doses administered andthe frequency of administration will be at the discretion and judgementof the physician treating the patient.

Example K is provided to illustrate a representative batch formula usedby the applicants to prepare capsules of Compound 1.

The pharmacological activity of the compounds of the present inventioncan be demonstrated by means of an in vitro screening model using aclone of NIH 3T3 cells transfected with the human bladder cancer T24(H-ras) oncogene. This cell line (PAP2) has been selected based upon itscharacterisation of a high level of ras expression and ras-dependentactivities which correlate with the high level of metastatic ability (S.A. Hill et al., in J. of the National Cancer Institute 1988; 80: 484-490and A. Chambers et. al. in Invasion and Metastasis 1990; 10: 225-240).The PAP2 cell line has been shown to have a ras-dependent increasedexpression of cathepsins, cysteine proteinases implicated in theprocesses of metastasis (A. Chambers et al., in Molecular Carcinogenesis1992; 5:238-245). Thus PAP2 cells exhibit functions which are relevantto the pathogenesis of human cancers. These cells were used for testingin vitro the effect of compounds on cell proliferation, proteolyticenzyme activity (metastasis) and apoptosis (programmed cell death). Wheninjected s.c. in immunodeficient (nude) mice these cells rapidly formsolid tumours and the anticancer activity of the tested compounds weremeasured in vivo after oral administration.

The results of a series of in vitro and in vivo tests led to thediscovery by the applicants that representative compounds of formula (I)and in particular Compound 1,

inhibit the growth of cancer cells in tissue culture,

induce apoptosis in cancer cells in tissue culture,

inhibit proteases in cancer cells which are involved in metastasis, and

demonstrate anti-cancer activity in nude mice bearing solid tumors. Theexperimental results presented in Tables 1-7b provide evidence thatcompounds of formula (I), and in particular Compound 1, are potentiallyuseful in the treatment of neoplastic diseases which include cancers ofthe hemopoietic and immune system, such as lymphomas and leukemias, aswell as cancers of the pancreas, colon, breast, thyroid, brain, lung,head and neck. This recently discovered anticancer activity of compoundsof formula (I) is unexpected and is independent of their previouslyreported activities as lipid lowering agents.

Results are expressed as mean±sem. Significance of difference wasestimated using the Student t-test for unpaired data.

EXAMPLE 1

INHIBITION OF RAS-DEPENDENT CELLULAR PROLIFERATION BY COMPOUNDS OFFORMULA (I)

A series of compounds of formula (I) were screened to determine the mostactive compounds and structure activity relationships. The inhibition ofPAP2 cell proliferation was selected as an initial screening test.

Briefly, PAP2 cells were seeded at a concentration of 3×10⁴ per well in24-well plates and were allowed to attach for 24 h. Test compounds wereadded at 10 and 20 μM final concentrations in 1% ethanol solutions.Cells were trypsinized after 48 h incubation and viable cells (excludingTrypan blue) were counted. The results obtained are listed in Tables 1 aand 1 b for the series of Compounds (Ia) and (Ib) respectively.

The compounds screened in this test were synthesized according to theprocedures described in the U.S. Pat. No. 5,043,330 (1991) correspondingto the European patent 0 339 237. Some examples (Examples A-J) areprovided to further illustrate the synthesis of novel derivativesaccording to the procedures described in the above-mentioned prior artdocuments.

                                      TABLE 1a                                    __________________________________________________________________________    Effect of phenol substituted gem-diphosphonates (Ia) on PAP2 Cells                                                      (Ia)                                 ##STR5##                                                                                                          Cell                                                                          (% count control)                        Cpd                                                                              X.sup.1                                                                           X.sup.2                                                                           X.sup.3                                                                          A  B Z.sup.1                                                                            Z.sup.2                                                                           Z.sup.3                                                                            Z.sup.4                                                                           10 μM                                                                          20 μM                             __________________________________________________________________________     1 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                                                             -70 -100                                  2 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H OH   OH  OH   OH  -31  -45                                  3 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H OMe  OMe OMe  OMe -21  -17                                  4 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H OEt  OEt OEt  OEt -30  -22                                  5 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H On-Pr                                                                              On-Pr                                                                             On-Pr                                                                              On-Pr                                                                             -22  -89                                  6 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H On-Bu                                                                              On-Bu                                                                             On-Bu                                                                              On-Bu                                                                             -26  -51                                  7 3-s-Bu                                                                            5-s-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H OEt  OEt OEt  OEt  -6  +3                                   8 3-s-Bu                                                                            5-s-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                                                             -21  -45                                  9 3-i-Pr                                                                            5-i-Pr                                                                            4-H                                                                              CH.sub.2                                                                         H OEt  OEt OEt  OEt -16  -31                                 10 3-i-Pr                                                                            5-i-Pr                                                                            4-H                                                                              CH.sub.2                                                                         H Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                                                             -20  -51                                 11 3-t-Bu                                                                            5-Me                                                                              4-H                                                                              CH.sub.2                                                                         H OEt  OEt OEt  OEt -14  -37                                 12 3-t-Bu                                                                            5-Me                                                                              4-H                                                                              CH.sub.2                                                                         H Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                                                              -3  -27                                 13 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              S  H OEt  OEt OEt  OEt -19  -39                                 14 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              S  H Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                                                             -53  -90                                 15 3-OMe                                                                             5-OMe                                                                             4-Me                                                                             CH.sub.2                                                                         H Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                                                             -23  -5                                  16 3-OEt                                                                             5-H 4-H                                                                              CH.sub.2                                                                         H Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                                                              -2  +5                                  17 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H OEt  OEt On-Bu                                                                              On-Bu                                                                              -6  -95                                 18 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H OEt  OEt Oi-Pr                                                                              Oi-Pr                                                                             -46  -59                                 19 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         Et                                                                              OEt  OEt OEt  OEt -21  -36                                 20 6-Cl                                                                               3,4-OCH.sub.2                                                                       CH.sub.2                                                                         H OEt  OEt OEt  OEt +20  -9                                  21 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         H   O(CH.sub.2).sub.3 O                                                                    O(CH.sub.2).sub.3 O                                                                  -32  -28                                 22 3-OMe                                                                             5-OMe                                                                             4-H                                                                              CH.sub.2                                                                         H OEt  OEt OEt  OEt                                          23 3-OMe                                                                             5-OMe                                                                             4-H                                                                              CH.sub.2                                                                         H Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                        24 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              CH.sub.2                                                                         Et                                                                              Oi-Pr                                                                              Oi-Pr                                                                             Oi-Pr                                                                              Oi-Pr                                                                             -25  -40                                 __________________________________________________________________________

                                      TABLE 1b                                    __________________________________________________________________________    Effect of phenol substituted gem-diphosphonates (Ib) on PAP2 Cells                                                   (Ib)                                    ##STR6##                                                                                                       Cell count                                                                    (% control)                                 Cpd                                                                              X.sup.1                                                                           X.sup.2                                                                           X.sup.3                                                                          k d Z.sup.1                                                                           Z.sup.2                                                                           Z.sup.3                                                                           Z.sup.4                                                                           10 μM                                                                          20 μM                                __________________________________________________________________________    25 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              0 0 OH  OH  OH  OH                                              26 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              0 0 OMe OMe OMe OMe -33 -17                                     27 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              0 0 OEt OEt OEt OEt -48 -45                                     28 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              0 0 Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             -28 -46                                     29 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              0 0 On-Pr                                                                             On-Pr                                                                             On-Pr                                                                             On-Pr                                                                             -51 -63                                     30 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              0 0 On-Bu                                                                             On-Bu                                                                             On-Bu                                                                             On-Bu                                                                             -13 -31                                     31 3-s-Bu                                                                            5-s-Bu                                                                            4-H                                                                              0 0 OEt OEt OEt OEt -30 -44                                     32 3-s-Bu                                                                            5-s-Bu                                                                            4-H                                                                              0 0 Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             -13 -54                                     33 3-i-Pr                                                                            5-i-Pr                                                                            4-H                                                                              0 0 OEt OEt OEt OEt  -5 +13                                     34 3-i-Pr                                                                            5-i-Pr                                                                            4-H                                                                              0 0 Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             -26 -62                                     35 3-t-Bu                                                                            5-Me                                                                              4-H                                                                              0 0 OEt OEt OEt OEt -14 -14                                     36 3-t-Bu                                                                            5-Me                                                                              4-H                                                                              0 0 Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             -24  -7                                     37 3-OMe                                                                             5-OMe                                                                             4-H                                                                              0 0 OEt OEt OEt OEt  -3 -28                                     38 3-OMe                                                                             5-OMe                                                                             4-H                                                                              0 0 Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                           39 3-OMe                                                                             5-OMe                                                                             4-Me                                                                             0 0 Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             -17 -34                                     40 3-OEt                                                                             5-H 4-H                                                                              0 0 Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             -30 -23                                     41 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              1 0 OEt OEt OEt OEt -40 -77                                     42 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              1 0 Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             Oi-Pr                                                                             -36 -71                                     43 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              0 0 OEt OEt On-Bu                                                                             On-Bu                                                                             -18 -54                                     44 3-t-Bu                                                                            5-t-Bu                                                                            4-H                                                                              0 0 OEt OEt Oi-Pr                                                                             Oi-Pr                                                                             -17 -28                                     45 3-t-Bu                                                                            5-t-Bu                                                                            4-Me                                                                             0 0 OEt OEt OEt OEt -10 -33                                     46 H    3,4-OCH.sub.2                                                                       0 0 OEt OEt OEt OEt -22 -26                                     47 H    3,4-(OCH.sub.2).sub.2                                                               0 0 OEt OEt OEt OEt -11 -30                                     __________________________________________________________________________

EXAMPLE 2

In vitro Results

INHIBITION OF RAS-DEPENDENT CELLULAR PROLIFERATION

Cell culture

H-Ras-transfected NIH 3T3 cells (PAP2) (S. A. Hill et al.; J Natl.Cancer Inst. 1988;80:484-490) were grown at 37° C. in a 5% CO₂atmosphere in Dulbecco's modified Eagle medium (DMEM) with 25 mM HEPESand 10% fetal calf serum. PAP2 cells were trypsinized and subculturedtwice a week prior confluency.

1. Inhibition of Cell proliferation

The effects of Compound 1 on cellular proliferation were monitored bytwo methods:

cell count with a hemacytometer and concurrent DNA determination

cell number estimation by calorimetric assay.

1.1. Cell count and DNA content

Briefly, PAP2 cells were seeded at a concentration of 3×10⁴ per dish in24-well plates 4 h before the addition of increasing concentrations ofthe tested compound. Cells were trypsinized at daily intervals. Analiquot of the cell suspension was counted with a hemacytometer. Theremaining cells were lysed in 0.01N NaOH and DNA concentration wasdetermined by spectrofluorimetry using 4,6-diamidino-2-phenylindole asfluorochrome and calf thymus DNA as standard.

                  TABLE 2a                                                        ______________________________________                                        Inhibition of the Proliferation of PAP2 Cells by Compound 1                   (cell number/well)                                                                   Compound 1 concentration                                                      0     0.1 μM                                                                             0.5 μM                                                                             1.0 μM                                                                           5.0 μM                                                                           10 μM                             ______________________________________                                        Cell nb/well                                                                           273750  256250  208750                                                                              186250                                                                              102500                                                                              71250                              sem      9437    7465    4270  15861 5951  8260                               % change         -6      -24   -32   -63   -74                                p                0.196   0.001 0003  0.001 0.001                              ______________________________________                                         The calculated IC50 value of Compound 1 for the inhibition of the growth      of PAP2 cells is 1.02 μM.                                             

                  TABLE 2b                                                        ______________________________________                                        Decrease in DNA Concentration Produced                                        by Compound 1 in Cultured PAP2 Cells                                          DNA (mg/well)                                                                        Compound 1 concentration                                                      0     0.1 μM                                                                             0.5 μM                                                                             1.0 μM                                                                           5.0 μM                                                                           10 μM                             ______________________________________                                        Cell nb/well                                                                           4.62    4.40    4.04  1.99  1.66  0.34                               sem      0.14    0.18    0.42  0.14  0.07  0.04                               % change         -5      -13   -57   -64   -93                                p                0.375   0.240 0.001 0.001 0.001                              ______________________________________                                         The calculated IC50 value of Compound 1 for decreasing the DNA content of     PAP2 cells is 2.77 μM.                                                

The results in Tables 2a and 2b show that compounds of formula (I), inparticular Compound 1, inhibit the growth of PAP2 cells in culture.

1.2. Colorimetric MTT assay

Cell number was estimated by the MTT assay performed essentially asdescribed by T. Mosmann in J. Immunol Method 1983;65:55-63. Briefly,PAP2 cells were seeded at a concentration of 1×10⁴ per dish in flat96-well plates (Falcon) 5 h prior the addition of the tested drugs.Following an incubation period of 24 h, 48 h or 72 h, 10 μl of MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]dissolved in PBS at 5 mg/ml were added to each well and incubated at 37°C. for 4 h. The medium was then removed and 100 μl of 0.04N HCl inisopropanol were added to the wells. Absorbance of the converted dye wasmeasured at 570 nm by a microplate reader with background substractionat 620 nm.

The calculated IC50 value of Compound 1 for the inhibition of theproliferation of PAP2 cells is 8.05 μM, as measured by the MTT method(after 72 h incubation). The results in Tables 3a, 3b and 3c confirm byanother assay that compounds of formula (I), in particular Compound 1,inhibit the growth of PAP2 cells in culture.

Effect of Compound 1 on the Proliferation of PAP2 Cells Measured by theMTT assay

                  TABLE 3a                                                        ______________________________________                                        Incubation with Compound 1 for 24 h                                           (OD at 570 nm)                                                                       Compound 1 concentration                                               0           1.0 μM                                                                             2.5 μM                                                                              1.5 μM                                                                           10 μM                                                                            25 μM                             ______________________________________                                        OD      39.9    39.5    37.6   35.9  28.9  21.4                               sem     2.1     4.6     1.2    1.0   1.1   1.0                                % change                                                                              0       -1      -6     -10   -28   -46                                p               0.942   0.370  0.109 0.0O1 0.001                              ______________________________________                                    

                  TABLE 3b                                                        ______________________________________                                        Incubation with Compound 1 for 48 h                                           (OD at 570 nm)                                                                       Compound 1 concentration                                                      0     1.0 μM                                                                             2.5 μM                                                                             5 μM                                                                             10 μM                                                                            25 μM                             ______________________________________                                        Mean     71.3    66.5    59.4  52.8  37.3  15.1                               sem      5.2     4.9     2.5   3.1   1.9   0.7                                % change 0       -7      -17   -26   -48   -79                                p                0.516   0.059 0.009 0.001 0.001                              ______________________________________                                    

                  TABLE 3c                                                        ______________________________________                                        Incubation with Compound 1 for 72 h                                           (OD at 570 nm)                                                                       Compound 1 concentration                                               0           1.0 μM                                                                             2.5 μM                                                                              5 μM                                                                             10 μM                                                                            25 μM                             ______________________________________                                        Mean    366.0   336.8   334.4  287.4 136.6 29.0                               sem     30.3    19.0    18.6   16.5  15.5  6.3                                % change        -8      -9     -21   -63   -92                                p               0.427   0.388  0.039 0.001 0.001                              ______________________________________                                    

3. Inhibition of DNA synthesis

DNA synthesis was measured by the incorporation of tritiated thymidineinto trichloroacetic acid (TCA) precipitable material by cultured PAP2cells. PAP2 cells were seeded in 24-well plates at 3×10⁴ cells per wellfor 2 days. Then following preincubation with Compound 1 dissolved inethanol, 0.5 μCi of methyl-[3H]-thymidine (specific activity 82 Ci/mmol)were added and labelling proceeded for 4 hours. The cells were thenwashed with 1 ml of cold phosphate-buffered saline (PBS), solubilizedwith 0.2 ml of 4% sodium dodecyl sulfate. The cellular extract wasprecipitated with 1 ml of 30% TCA, and kept for 1 h on ice. Theprecipitates were collected by filtration onto fiber glass filters andwashed with 5 ml of 5% TCA. Radioactivity on the filters was counted ina liquid scintillation counter.

                  TABLE 4                                                         ______________________________________                                        Inhibition of DNA Synthesis by Compound 1                                     in ras Transfected Cells (PAP2)                                               3H-Thymidine incorporation into DNA                                           (cpm)                                                                                Compound 1 concentration                                               0           0.5 μM                                                                             2 μM  5 μM                                                                             10 μM                                                                            25 μM                             ______________________________________                                        cpm     53021   45607   37130  30226 18772 14100                              sem     7296    5354    2590   3945  1481  2766                               % change                                                                              0       -14     -30    -43   -65   -73                                p               0.427   0.075  0.016 0.009 0.005                              ______________________________________                                         The calculated IC50 value of Compound 1 for the inhibition DNA synthesis      in PAP2 cells is 3.75 μM.                                             

Results in Table 4 show that compounds of formula (I), in particularCompound 1, inhibit DNA synthesis in ras transfected cells.

4. Induction of apoptosis

Compounds of formula (I) have also been shown to have a selective effectin stimulating cell death in the PAP2 cell line at 5 μM or higherconcentrations. This specific effect in selectively stimulatingprogrammed cell death in cancer cells was verified in two other humancancer cell lines (HepG2 and SW480) with Compound 1 indicating thatthese compounds not only inhibit cell growth but in addition producecancer cell self-destruction (Table 5). This has been confirmed by gelelectrophoresis showing DNA fragmentation, the hallmark of apoptosis.

                  TABLE 5                                                         ______________________________________                                        The Effect of Compound 1 on Cell Viability in                                 Human Cancer Cell Lines                                                                 Nb of Viable                                                                              Nb of Viable                                                      Cells at    Cells at                                                Cell line 0 hrs       24 hrs      % Change                                    ______________________________________                                        SW 480    38'800      0           -100.0%                                     HepG2     214'700     82'000      -61.8%                                      ______________________________________                                         Cells are cultured in 24well plates for 48 h prior treatment.                 Treatments are 10 μM compounds for 24 h.                                   Counting: cells are trypsinized and viable cells are counted.            

5. Effect of Compound 1 on a wide range of human cancer cell lines

Compound 1 was tested for its anti-proliferative activity in a widerange of established human derived cancer cell lines to confirm thepotential use of these compounds in relevant human cancers (Table 6).The results indicate that Compound 1 is effective in decreasing cellproliferation in greater than 90% of the more than 50 human cancer celllines tested. Compounds of formula (I) therefore can be considereduseful in treating a wide range of human cancers with and withoutras-mutations.

                  TABLE 6                                                         ______________________________________                                        Anti-Proliferative Effects Compound 1 on Human Tumor Derived                  Cell Lines                                                                                                   Inhibition of                                                                 Proliferation                                  Tumor Type      Cell line      (% Control)                                    ______________________________________                                        Leukemia        CCRF-CEM       -61.8                                          Leukemia        HL-60 (tb)     -95.3                                          Leukemia        K-562          -61.2                                          Non-Small Cell Lung Cancer                                                                    A549/ATCC      -55.0                                          Non-Small Cell Lung Cancer                                                                    EKVX           -27.0                                          Non-Small Cell Lung Cancer                                                                    HOP-62         -13.8                                          Non-Small Cell Lung Cancer                                                                    HOP-92         -85.1                                          Non-Small Cell Lung Cancer                                                                    NCI-H23        -21.8                                          Non-Small Cell Lung Cancer                                                                    NCI-H322M      -12.5                                          Non-Small Cell Lung Cancer                                                                    NCI-H460       -40.7                                          Non-Small Cell Lung Cancer                                                                    NCI-H522       -59.8                                          Colon Cancer    COLO 205       -55.9                                          Colon Cancer    HCC-2998       -50.7                                          Colon Cancer    HCT-116        -68.7                                          Colon Cancer    HCT-15         -19.5                                          Colon Cancer    HT29           -83.0                                          Colon Cancer    KM12           -32.5                                          Colon Cancer    SW-620         -21.4                                          CNS Cancer      SF-268         -21.2                                          CNS Cancer      SF-295         -49.6                                          CNS Cancer      SF-539         +1.5                                           CNS Cancer      SNB-19         -55.8                                          CNS Cancer      SNB-75         -54.4                                          CNS Cancer      U251           -41.6                                          Melanoma        LOX IMVI       -18.7                                          Melanoma        MALME-3M       -20.4                                          Melanoma        M14            -66.7                                          Melanoma        SK-MEL-2       -6.1                                           Melanoma        SK-MEL-28      -25.0                                          Melanoma        SK-MEL-5       -51.8                                          Melanoma        UACC-257       -26.9                                          Melanoma        UACC-62        -30.9                                          Ovarian Cancer  IGROV1         -24.4                                          Ovarian Cancer  OVCAR-3        -47.3                                          Ovarian Cancer  OVCAR-4        -18.9                                          Ovarian Cancer  OVCAR-5        -17.6                                          Ovarian Cancer  OVCAR-8        -37.6                                          Ovarian Cancer  SK-OV-3        -28.1                                          Renal Cancer    786-0          -28.7                                          Renal Cancer    ACHN           -43.4                                          Renal Cancer    CAKI-1         -31.1                                          Renal Cancer    SN12C          -1.0                                           Renal Cancer    TK-10          -18.6                                          Renal Cancer    UO-31          -35.9                                          Prostate Cancer PC-3           -75.9                                          Prostate Cancer DU-145         -21.3                                          Breast Cancer   MCF-7          -57.0                                          Breast Cancer   MCF-7/ADR-RES  -49.3                                          Breast Cancer   MDA-MB-231/ATCC                                                                              -50.9                                          Breast Cancer   MDA-MB-435     -51.6                                          Breast Cancer   MDA-N          -43.3                                          Breast Cancer   BT-549         -51.3                                          Breast Cancer   T-47D          -62.1                                          ______________________________________                                    

EXAMPLE 2

In vivo Results

INHIBITION OF TUMOUR GROWTH

Seven to nine-week old female nude mice (Swiss nu/nu) were injected s.c.with 0.5 ml of PAP2 cells (5×10⁵ cells per 0.5 ml of DMEM) on day 0.Oral treatment with Compound 1 (50 mg/kg) started on the same day. Thetreated group (n=1 8) received Compound I mixed with food (0.03% w/w)and the control group (n=22) received a diet without added compound. Atday 15, the mice were weighed, sacrificed and the tumors were excisedand weighed.

                  TABLE 7a                                                        ______________________________________                                        Effect of Oral Treatment with Compound 1 (50 mg/kg)                           on Tumor Weight Produced by s.c. Injection of PAP2 cells in Nude Mice                      Body Weight (g)                                                                             Tumor Weight (g)                                   Animal Group (mean ± sem)                                                                             (mean ± sem)                                    ______________________________________                                        Control      24.2 ± 0.5 0.228 ± 0.041                                   n = 21)                                                                       Treated      24.6 ± 0.7 0.053 ± 0.016                                   (n = 18)                                                                      % change     +2            -77                                                p            0.6470        0.0006                                             ______________________________________                                    

The results in Table 7a show that the mean tumor weight in the treatmentgroup decreased significantly, indicating that Compound 1 is a potentantitumor agent. Mice body weight in both control and treated groups areidentical, thus establishing the lack of toxicity of Compound 1.

In another set of experiments Compound 1 was tested at doses of 12.5, 50and 100 mg/kg. The results in Table 7b show that Compound 1 inhibitstumor growth in nude mice at doses as low as 12.5 mg/kg and in a dosedependent manner. These results demonstrate that compounds of formula(I), in particular Compound 1, are potent orally active antitumorcompounds which do not have a toxic effect on normal cells, tissues ororgans.

                  TABLE 7b                                                        ______________________________________                                        Decrease in Tumour Weight by Oral Treatment                                   of Nude Mice Injected with PAP2 Cells                                         with Different Doses of Compound 1                                                           Body Weight (g)                                                                            Tumor Weight (g)                                  Animal Group   (mean ± sem)                                                                            (mean ± sem)                                   ______________________________________                                        Control (n = 14)                                                                             24.7 ± 0.6                                                                              0.276 ± 0.056                                  Treated, 12.5 mg/kg (n = 5)                                                                  23.9 ± 0.8                                                                              0.053 ± 0.026                                  % change       -3           -81                                               p              0.4562       0.0472                                            Treated, 50 mg/kg (n = 6)                                                                    27.2 ± 1.0                                                                              0.037 ± 0.020                                  % change       +10          -87                                               p              0.0523       0.0211                                            Treated, 100 mg/kg (n = 6)                                                                   24.5 ± 0.3                                                                              0.025 ± 0.01 1                                 % change       -1           -91                                               p              0.7619       0.0151                                            ______________________________________                                    

EXAMPLE A

Tetraisopropyl2-(3,5-diisopropyl4-hydroxyphenyl)-ethenylidene-1,1-diphosphonate##STR7##

Titanium tetrachloride (13.83 g, 73 mmol) was added dropwise to dry THF(80 ml) maintained at 0° C. The resulting mixture was treatedsequentially at 0° C. with 3,5-diisopropyl-4-hydroxybenzaldehyde (5.0 g,24 mmol), tetraisopropyl methylenediphosphonate (10.85 g, 32 mmol) andN-methylmorpholine (14.71 g, 146 mmol). The reaction mixture was stirredfor 12 h at room temperature and 80 ml water were added. The quenchedreaction mixture was extracted with diethylether (3×60 ml), the combinedether fractions were extracted with a saturated NaCl solution until theaqueous washes had a neutral pH. After drying over MgSO₄, the organicsolvent was evaporated and the residue was purified by columnchromatography on silica using as eluant a mixture of CH₂ Cl₂ :MeOH(95:5) to give 8 g (62%) of a solid; mp=87-88° C.

MS: m/e=532: M⁺, 367: M⁺ --PO₃ iPr₂

NMR (CDCl₃)

δ=8.22 (dd, J=31 and 48 Hz, 1H): Ph--CH═C--P₂

7.7 (s, 2H): aromatic H

5.6 (s, 1H): OH,

4.85-4.63 (2m, 4H): P--O--CHMe₂

3.16 (septet, 2H): Ph--CHMe₂

1.39, 1.36, 1.27, 1.23 and 1.16 (8d, 36H total): P--O--CHMe₂ andPh--CHMe₂

EXAMPLE B

Tetraisopropyl2-(3,5-diisopropyl-4-hydroxyphenyl)-ethylidene-1,1-diohosphonate##STR8## Tetraisopropyl2-(3,5-diisopropyl-4-hydroxyphenyl)-ethenylidene-1,1-diphosphonate (5 g,9.4 mmol) was dissolved in ethanol (50 ml) and the solution washydrogenated for 4 h over 2 g of 10% palladium on carbon at 50 psi atroom temperature. The catalyst was filtered, the solvent was evaporatedand the residue was purified by column chromatography on silica using aseluant a mixture of CH₂ Cl₂ :MeOH (95:5) to give 2.5 g (50%) of a solid;mp=90-91° C.

MS: m/e =534: M⁺, 369: M⁺ --PO₃ iPr₂

NMR (CDCl₃)

δ=6.94 (m, 2H) : aromatic H,

4.8-4.7 (m, 5H): P--O--CHMe₂ and OH

3.2-3.1 (several m, 6H total ): Ph--CH₂ --CH and Ph--CHMe₂

2.51 (tt, J=6 and 24H, 1H): Ph--CH₂ --CH

1.33-1.21 (several d, 36H total): P--O--CHMe₂ and Ph--CHMe₂

EXAMPLE C

Tetraisopropyl 2-(3,4,5-trimethoxyphenyl)-ethenylidene-1,1-diphosphonate##STR9## 3,4,5-Trimethoxybenzaidehyde (7 g, 35.7 mmol) was treated withtitanium tetrachloride, tetraisopropyl methylenediphosphonate andN-methylmorpholine in THF as described in Example A to give 11.5 g (62%)of the title compound.

MS: m/e=522: M⁺, 357: M⁺ --PO₃ iPr₂

NMR (CDCl₃)

δ=8.21 (dd, J=30 and 48 Hz, 1H): Ph--C H═C--P₂

7.28: (s, 2H): aromatic H

4.85-4.63 (2m, 4H): P--O--CHMe₂

3.9 (t, 9H): Ph--OMe

1.4, 1.36 and 1.22 (4d, 24H total): P--O--CHMe₂

EXAMPLE D

Tetraisopropyl 2-(3,4,5-trimethoxyphenyl)-ethylidene-1,1-diphosphonate##STR10## Tetraisopropyl2-(3,4,5-trimethoxyphenyl)-ethenylidene-1,1-diphosphonate (7 g, 13.4mmol) was hydrogenated over 10% Pd/C as described in Example B to give5.7 g (81%) of the title compound.

MS: m/e=524: M⁺, 359: M⁺ --PO₃ iPr₂

NMR (CDCl₃)

δ=6.55 (s, 2H): aromatic H,

4.8-4.7 (m, 4H): P--O--CHMe₂

3.85 and 3.81 (2s, 9H): Ph--OMe

3.17 (dt, J=6 and 16 Hz, 2H): Ph--CH₂ --CH

2.50 (tt, J=6 and 24 Hz, 1H): Ph--CH₂ --CH

1.33-1.26 (several d, 24H total): P--O--CHMe₂

EXAMPLE E

Tetraisopropyl2-(3-tert-butyl-4-hydroxy-5-methylphenyl)-ethenylidene-1,1-diphosphonate##STR11## 3-Tert-butyl-4-hydroxy-5-methylbenzaldehyde (6 g, 31.3 mmol)was treated with titanium tetrachloride, tetraisopropylmethylenediphosphonate and N-methylmorpholine in THF as described inExample A to give 4.2 g (62%) of the title compound.

MS: m/e=518: M⁺, 353: M⁺ --PO₃ iPr₂

NMR (CDCl₃)

δ=8.19 (dd, J=30 and 48 Hz, 1H): Ph--CH═C--P₂

7.71-7.67 (m, 2H) aromatic H,

5.6 (s, 1H): OH,

4.8-4.7 (2m, 4H): P--O--CHMe₂

2.26 (s, 3H): Ph--Me

1.40 (s, 9H): Ph--t--Bu

1.38, 1.36, 1.24 and 1.20 (8d, 24H total): P--O--CHMe₂

EXAMPLE F

Tetraisopropyl2-(3-tert-butyl-4-hydroxy-5-methylphenyl)-ethylidene-1,1-diphosphonate##STR12## Tetraisopropyl2-(3-tert-butyl-4-hydroxy-5-methylphenyl)-ethenylidene-1,1-diphosphonate(5 g, 9.6 mmol) was hydrogenated over 10% Pd/C as described in Example Bto give 2.9 g (58%) of the title compound.

MS: m/e=520: M⁺, 355: M⁺ --PO₃ iPr2

NMR (CDCl₃)

δ=7.02 and 6.92 (2m, 2H): aromatic H,

4.8-4.7 (m, 5H): P--O--CHMe₂ and OH

3.11 (dt, J=6 and 17 Hz, 2H): Ph--CH₂ --CH

2.49 (tt, J=6 and 24 Hz, 1H): Ph--CH₂ --CH

2.21 (s, 3H): Ph--Me

1.39 (s, 9H): Ph--t--Bu

1.31, 1.26 and 1.24(3d, 24H): P--O--CHMe₂

EXAMPLE G

Tetraisopropyl2-(3-ethoxy-4-hydroxyphenyl)-ethenylidene-1,1-diphosphonate ##STR13##3-Ethoxy-4-hydroxybenzaldehyde (6 g, 36.1 mmol) was treated withtitanium tetrachloride, tetraisopropyl methylenediphosphonate andN-methylmorpholine in THF as described in Example A to give 4.2 g (62%)of the title compound, mp=143-144° C.

MS: m/e=492: M⁺, 327: M⁺ --PO₃ iPr₂

NMR (CDCl₃)

δ=8.19(dd, J=31 and 48 Hz, 1H): Ph--CH═C--P₂

7.9, 7.3 and 6.91(3m, 3H): aromatic H,

6.2 (s, 1H) : OH

4.85-4.63 (2m, 4H): P--O--CHMe₂

4.19 (q, J=7 Hz): Ph--OCH₂ --CH₃

1.46 (t, J=7 Hz): Ph--OCH₂ --CH₃

1.39, 1.36, 1.23 and 1.21 (4d, 24H total): Ph--CHMe₂

EXAMPLE H

Tetraisoproyl 2-(3-ethoxy-4-hydroxy-phenyl)-ethylidene-1,1-diphosphonate##STR14## Tetraisopropyl2-(3-ethoxy-4-hydroxyphenyl)-ethenylidene-1,1-diphosphonate (5 g, 10.16mmol) was hydrogenated over 10% Pd/C as described in Example B to give4.5 g (90%) of the title compound.

MS: m/e=494: M⁺, 329: M⁺ --PO₃ iPr₂

NMR (CDCl₃)

δ=6.85-6.75 (several m, 3H): aromatic H

5.65 (s, 1H): OH

4.8-4.7 (m, 4H): P--O--CHMe₂

4.10 (q, J=7 Hz): Ph--OCH₂ --CH₃

3.14 (dt, J=6 and 16 Hz, 2H): Ph--CH₂ --CH

2.45 (tt, J=6 and 24 Hz, 1H): Ph--CH₂ --CH

1.43 (t, J=7 Hz): Ph--OCH₂ --CH₃

1.32-1.25 (4 partially overlapping d, 24H total): P--O--CHMe₂

EXAMPLE I

Tetraethyl2-(3,5-di-tert-butyl-4-methoxyphenyl)-ethenylidene-1,1-diphosphonate##STR15## 3,5-Di-tert-butyl-4-methoxybenzaldehyde (2.0 g, 8.1 mmol) wastreated with titanium tetrachloride, tetraethyl methylenediphosphonateand N-methylmorpholine in THF as described in Example A to give 3.0 g(72%) of the title compound, mp=66-67° C.

MS: m/e=518: M⁺, 381: M⁺ --PO₃ Et₂

NMR (CDCl₃)

δ=8.26 (dd, J=30 and 48 Hz, 1H): Ph--CH═C--P₂

7.8 (s, 2H): aromatic H,

4.25-4.00 (2m, 8H): P--O--CH₂ --CH₃

3.69 (s, 3H): Ph--OMe

1.44 (s, 18H): Ph--t--Bu

1.38 and 1.70 (2t, 12H): Ph--OCH₂ --CH₃

EXAMPLE J

Tetraisopropyl1-(3,5-di-tert-butyl-4-hydroxyphenyl)-butylidene-2,2-diphosphonate##STR16## Tetraisopropyl propylidene-1,1-diphosphonate was prepared byreacting tetraisopropyl methylenediphosphonate with 3 equivalents ofethyl iodide in presence of NaH in THF.

Tetraisopropyl propylidene-1,1-diphosphonate (2.2 g, 6.0 mmol) was addedto a suspension of 60% NaH (0.5 g, 12.0 mmol) in 20 ml of dry THF andthe mixture was stirred until the NaH disappeared.3,5-di-tert-butyl-4-hydroxybenzylchloride (1.5 g, 6 mmol) in 10 ml THFwas added and the mixture was refluxed overnight. After work up, columnchromatography on silica using CHCl₃ : AcOEt (8:2) as eluant gave 1.5 g(42%) of the title compound.

MS: m/e=590: M⁺, 425: M⁺ --PO₃ ^(i) Pr₂, base Peak 341: M⁺ -2× propen

mp=131-132° C.

EXAMPLE K

Typical Example of Compound Formulation

Active Component

Compound 1

Inactive Component

Pregelatinazed Starch NF

Size 3 opaque dark blue gelatin capsules

    ______________________________________                                        Representative Batch Formula                                                  Typical batch size 2000 capsules                                                            g/batch                                                         Ingredients     1 mg       10 mg  50 mg                                       ______________________________________                                        Compound 1      2.00       20.0   100.0                                       Pregelatinized starch NF                                                                      431.6      408.8  291.0                                       Total           433.6      428.8  391.0                                       ______________________________________                                    

What is claimed is:
 1. A method of treating or preventing a neoplasticdisease in a patient, wherein the neoplastic disease is chosen fromcancers of the hemopoietic and immune system and cancers of thepancreas, colon, breast, thyroid, brain, lung, head and neck, saidmethod comprising administering to a patient in need of such therapy, atherapeutically effective amount of a compound represented by formula I##STR17## wherein: Z¹, Z², Z³ and Z⁴ are each --OR wherein R isindependently chosen from --H and straight or branched alkyl comprisingfrom 1 to 8 carbon atoms;X¹ and X² are independently straight orbranched alkyl comprising from 1 to 8 carbon atoms; X³ is --H; A is--(CH₂)_(n) --, wherein n is an integer from 1 to 7; B is chosen from--H and alkyl comprising from 1 to 4 carbon atoms; and t is 1; or apharmaceutically acceptable salt thereof.
 2. The method according toclaim 1, wherein Z¹, Z², Z³ and Z⁴ are each independently chosen fromhydroxy, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butyloxy,sec-butyloxy, and tert-butyloxy.
 3. The method according to claim 2,wherein Z¹, Z², Z³ and Z⁴ are the same.
 4. The method according to claim3, wherein Z¹, Z², Z³ and Z⁴ are each isopropyloxy.
 5. The methodaccording to claim 1, wherein X¹ and X² are independently straight orbranched alkyl comprising from 1 to 5 carbon atoms.
 6. The methodaccording to claim 5, wherein X¹ and X² are independently chosen frommethyl, ethyl, n-propyl, isopropyl, s-butyl and t-butyl.
 7. The methodaccording to claim 6, wherein X¹ and X² are the same.
 8. The methodaccording to claim 7, wherein X¹ and X² are each t-butyl.
 9. The methodaccording to claim 1, wherein the compound is tetraisopropyl2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate. 10.The method according to claim 1, wherein the compound is chosenfrom:2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonicacid; tetramethyl2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate;tetraethyl2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate;tetra-n-propyl2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate;tetra-n-butyl2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate;tetraisopropyl2-(3,5-di-sec-butyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate;tetraethyl 2-(3,5-diisopropyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate;tetraisopropyl2-(3,5-diisopropyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate;tetraethyl2-(3-tert-butyl-4-hydroxy-5-methylphenyl)-ethylidene-1,1-diphosphonate;tetraisopropyl2-(3-tert-butyl-4-hydroxy-5-methylphenyl)-ethylidene-1,1-diphosphonate;tetraethyl1-(3,5-di-tert-butyl-4-hydroxyphenyl)-butylidene-2,2-diphosphonate; andtetraisopropyl1-(3,5-di-tert-butyl-4-hydroxyphenyl)-butylidene-2,2-diphosphonate. 11.The method according to claim 1 wherein the neoplastic disease is a rasdependent cancer.
 12. The method according to claim 1 wherein theneoplastic disease is chosen from lymphomas and leukemias.
 13. A methodof preventing transformation of a normal cell of a patient into a tumorcell by mutated ras activities, wherein said tumor cell is chosen fromcancers of the hemopoietic and immune system and cancers of thepancreas, colon, breast, thyroid, brain, lung, head and neck, saidmethod comprising treating a normal cell of a patient in need of suchtreatment, with an amount of a compound effective to block said mutatedras activities, said compound represented by formula I ##STR18##wherein: Z¹, Z², Z³ and Z⁴ are each --OR wherein R is independentlychosen from --H and straight or branched alkyl comprising from 1 to 8carbon atoms;X¹ and X² are independently straight or branched alkylcomprising from 1 to 8 carbon atoms; X³ is --H; A is --(CH₂)_(n) --,wherein n is an integer from 1 to 7; B is chosen from --H and alkylcomprising from 1 to 4 carbon atoms; and t is 1; or a pharmaceuticallyacceptable salt thereof.
 14. The method according to claim 13, whereinthe compound is tetraisopropyl2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethylidene-1,1-diphosphonate. 15.A method of treating or preventing a neoplastic disease in a patient,wherein the neoplastic disease is chosen from leukemia, non-small celllung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renalcancer, prostrate cancer and breast cancer, said method comprisingadministering to a patient in need of such therapy, a therapeuticallyeffective amount of a compound represented by formula I ##STR19##wherein: Z¹, Z², Z³ and Z⁴ are each --OR wherein R is independentlychosen from --H and straight or branched alkyl comprising from 1 to 8carbon atoms;X¹ and X² are independently straight or branched alkylcomprising from 1 to 8 carbon atoms; X³ is --H; A is --(CH₂)_(n) --,wherein n is an integer from 1 to 7; B is chosen from --H and alkylcomprising from 1 to 4 carbon atoms; and t is 1; or a pharmaceuticallyacceptable salt thereof.
 16. A method of treating or preventing aneoplastic disease in a patient, wherein the neoplastic disease ischosen from cancers of the kidney and liver, said method comprisingadministering to a patient in need of such therapy, a therapeuticallyeffective amount of a compound represented by formula I ##STR20##wherein: Z¹, Z², Z³ and Z⁴ are each --OR wherein R is independentlychosen from --H and straight or branched alkyl comprising from 1 to 8carbon atoms;X¹ and X² are independently straight or branched alkylcomprising from 1 to 8 carbon atoms; X³ is --H; A is --(CH₂)_(n) --,wherein n is an integer from 1 to 7; B is chosen from --H and alkylcomprising from 1 to 4 carbon atoms; and t is 1; or a pharmaceuticallyacceptable salt thereof.